Non-ribosomal peptide synthetases (NRPSs) are enzymatic assembly lines that produce a wealth of natural products in bacteria and fungi. These products confer virulence to pathogens and often are valuable therapeutics, including antibiotics (penicillin, bacitracin), antitumor agents (bleomycin, epothilone), and immunosuppressants (rapamycin). NRPSs use multiple domains, organized in contiguous modules, to covalently load, modify, and join substrates in an assembly line fashion. This remarkable organization holds the promise of producing novel pharmaceuticals by swapping domains or modules to reprogram the NRPS assembly line. However, most NRPS domain interactions remain uncharacterized, the structure and mechanism of important domains are unknown, and artificially engineered NRPSs are generally unproductive. This proposal aims to reveal the structural basis for heterocycle formation and alteration of their stereochemistry in NRPSs, and unravel domain communication during related synthesis. We will focus on HMWP2, an NRPS that participates in the synthesis of yersiniabactin (Ybt), a virulence factor found in pathogens such as Yersinia pestis, the causative agent of the bubonic plague, Y. enterocolitica, a food pathogen, and uropathogenic E. coli, responsible for urinary tract infections. Our results will contribute to understanding the molecular logic employed by these pathogens during infections. We will primarily use Nuclear Magnetic Resonance (NMR) because of the transient nature of molecular interactions, as well as the existence of multiple conformers in equilibrium. NMR will be used to determine the structures of cyclization and epimerization domains, identify binding sites of chemical and protein substrates, and characterize dynamics within domains during molecular interactions. In a synergistic approach, we will combine mutagenesis and biochemical assays with NMR experiments to provide an atomic level description of reaction mechanisms. The size of the multi-domain complexes reaches 70 kDa and is a challenge for NMR studies, which are typically limited to 20 kDa. In the past we designed methods to solve structures of 50 kDa proteins and obtain useful data from 800kDa complexes. HMWP2 will provide a model system to further develop new NMR methods for large dynamic proteins and to understand conformational rearrangements during protein interactions in general. Our research will simultaneously enable us to push the frontier in NMR studies of larger proteins, help understand the function of protein dynamics in biological systems, reveal the structure of critical domains, and provide a basis for efficient reprogramming of NRPS assembly lines to produce new pharmaceuticals.